NK-cell receptor modulation in viral infections.

Natural killer (NK) cells play a crucial role in controlling viral infections. The ability to kill infected cells without prior immunization, yet being tolerant to self, healthy cells, depends on the balance of germ-line encoded surface receptors. NK-cell receptors are divided into either activating, leading to activation of NK cell and its cytotoxic and pro-inflammatory activity, or inhibitory, providing tolerance for a target cell. The signals from inhibitory receptors dominate and NK-cell activation requires stimulation of activating receptors. In viral infections, NK-cell interaction with infected cells can result in activation, memory-like NK-cell differentiation, or NK-cell exhaustion, which constitutes one of the viral immune evasion mechanisms. All of these states are associated with the modulation of NK-cell receptor expression. In this review, we summarize the current knowledge of NK-cell receptors and their role in viral infection control, as well as the alterations of their expression observed in acute or chronic infections. We present recently discovered SARS-CoV-2-mediated modulation of NK-cell receptor expression and compare them with other human viral infections. Finally, since modulation of NK-cell receptor activation gives a promising addition to currently used antiviral therapies, we briefly discuss the clinical significance and future perspective of the application of agonists or antagonists of activating and inhibitory receptors, respectively. In sum, our review shows that although much is known about NK-cell receptor biology, a deeper understanding of NK-cell receptors role in viral infections is still needed.

Clinical and experimental treatment of primary humoral immunodeficiencies.

Selective IgA deficiency (sIgAD), common variable immunodeficiency (CVID), and transient hypogammaglobulinemia of infancy (THI) are the most frequent forms of primary antibody deficiencies. Difficulties in initial diagnosis, especially in the early childhood, the familiar occurrence of these diseases, as well as the possibility of progression to each other suggest common cellular and molecular patomechanism and a similar genetic background. In this review, we discuss both similarities and differences of these three humoral immunodeficiencies, focusing on current and novel therapeutic approaches. We summarize immunoglobulin substitution, antibiotic prophylaxis, treatment of autoimmune diseases, and other common complications, i.e. cytopenias, gastrointestinal complications, and granulomatous disease. We discuss novel therapeutic approaches such as allogenic stem cell transplantation and therapies targeting-specific proteins, dependent on the patient's genetic defect. The diversity of possible therapeutics models results from a great heterogeneity of the disease variants, implying the need of personalized medicine approach as a future of primary humoral immunodeficiencies treatment.

N-methylmorpholine incorporation into the structure of biphenyl leads to the bioactive inhibitor of PD-1/PD-L1 interaction.

We present new small-molecular probes targeting the human PD-L1 protein. The molecules were designed by incorporating a newly discovered N-methylmorpholine substituent into a known biphenyl-based structure. Four prototype derivatives of 4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-carbonitrile (STD4), comprising a morpholine substituent fused with a biphenyl core at different orientations were first verified for their potential binding to PD-L1 using the molecular docking method. A more favorable 7-phenyl derivative of STD4 was then equipped with an amide bond, pyridine, and either a tris(hydroxymethyl)aminomethane or serinol tail leading to two final molecules. Among them, compound 1c showed activity in three bioassays, i.e., the homogenous time-resolved fluorescence (HTRF) assay, immune checkpoint blockade (ICB) assay, and T-cell activation (TCA) assay. Our work shows that morpholine can substitute for dioxane and becomes a promising component in PD-L1-targeting molecules. This finding unlocks new avenues for optimizing PD-L1-targeting compounds, presenting exciting prospects for future developments in this field.

TGF-ß Signalling Regulates Cytokine Production in Inflammatory Cardiac Macrophages during Experimental Autoimmune Myocarditis.

Myocarditis is characterized by an influx of inflammatory cells, predominantly of myeloid lineage. The progression of myocarditis to a dilated cardiomyopathy is markedly influenced by TGF-ß signalling. Here, we investigate the role of TGF-ß signalling in inflammatory cardiac macrophages in the development of myocarditis and post-inflammatory fibrosis. Experimental autoimmune myocarditis (EAM) was induced in the LysM-Cre × R26-stop-EYFP × Tgfbr2-fl/fl transgenic mice showing impaired TGF-ß signalling in the myeloid lineage and the LysM-Cre × R26-stop-EYFP control mice. In EAM, immunization led to acute myocarditis on day 21, followed by cardiac fibrosis on day 40. Both strains showed a similar severity of myocarditis and the extent of cardiac fibrosis. On day 21 of EAM, an increase in cardiac inflammatory macrophages was observed in both strains. These cells were sorted and analysed for differential gene expression using whole-genome transcriptomics. The analysis revealed activation and regulation of the inflammatory response, particularly the production of both pro-inflammatory and anti-inflammatory cytokines and cytokine receptors as TGF-ß-dependent processes. The analysis of selected cytokines produced by bone marrow-derived macrophages confirmed their suppressed secretion. In conclusion, our findings highlight the regulatory role of TGF-ß signalling in cytokine production within inflammatory cardiac macrophages during myocarditis.

Structural and biological characterization of pAC65, a macrocyclic peptide that blocks PD-L1 with equivalent potency to the FDA-approved antibodies.

Recent advances in immuno-oncology have opened up new and impressive treatment options for cancer. Notwithstanding, overcoming the limitations of the current FDA-approved therapies with monoclonal antibodies (mAbs) that block the PD-1/PD-L1 pathway continues to lead to the testing of multiple approaches and optimizations. Recently, a series of macrocyclic peptides have been developed that exhibit binding strengths to PD-L1 ranging from sub-micromolar to micromolar. In this study, we present the most potent non-antibody-based PD-1/PD-L1 interaction inhibitor reported to date. The structural and biological characterization of this macrocyclic PD-L1 targeting peptide provides the rationale for inhibition of both PD-1/PD-L1 and CD80/PD-L1 complexes. The IC50 and EC50 values obtained in PD-L1 binding assays indicate that the pAC65 peptide has potency equivalent to the current FDA-approved mAbs and may have similar activity to the BMS986189 peptide, which entered the clinical trial and has favorable safety and pharmacokinetic data. The data presented here delineate the generation of similar peptides with improved biological activities and applications not only in the field of cancer immunotherapy but also in other disorders related to the immune system.

Transcriptome profiling of regulatory T cells from children with transient hypogammaglobulinemia of infancy.

Transient hypogammaglobulinemia of infancy (THI) is one of the most common forms of hypogammaglobulinemia in the early childhood. THI is usually associated with chronic, recurrent bacterial and viral infections, life-threatening in some cases, yet its pathogenesis is still largely unknown. As our previous findings indicated the possible role of Treg cells in the pathomechanism of THI, the aim of the current study was to investigate gene expression profile of Treg cells isolated from THI patients. The transcriptome-wide gene profiling was performed using microarray technology on THI patients in two time-points: during (THI-1), and in resolution phase (THI-2) of hypogammaglobulinemia. As a result, a total of 1086 genes were differentially expressed in THI-1 patients, when compared to THI-2 as well as control group. Among them, 931 were up- and 155 downregulated, and part of them encodes genes important for Treg lymphocyte biology and function, i.e. transcription factors/cofactors that regulate FOXP3 expression. Thus, we postulate that Treg cells isolated from THI patients during hypogammaglobulinemia display enhanced suppressor transcriptome signature. Treg expression profile of THI children after normalization of Ig levels largely resembles the results obtained in healthy control group, suggesting THI Treg transcriptome seems to return to that observed in healthy children. Taken together, we suggest that THI pathomechanism is associated not only with transiently elevated Treg cell numbers, but also with their enhanced regulatory/inhibitory functions. These findings expand our knowledge of human Treg cells and may be useful for the future diagnosis or management of THI.

Serum concentration of dickkopf-related protein 1 (DKK1) in psoriatic arthritis in the context of bone remodelling.

Psoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by the pathological occurrence of two opposite phenomena-osteoresorption and osteogenesis. Dickkopf-related protein 1 (DKK1) which inhibits the Wingless protein (Wnt) signalling pathway has been shown to be a master regulator of bone remodeling in inflammatory rheumatic diseases. However, the exact relationship between DKK1 serum level and bone remodelling is not clear. The goal of this study is to review state-of-the-art knowledge on the association of serum DKK1 with a bone remodelling in PsA. The MEDLINE-PubMed, EMBASE, Scopus, Web of Science and DOAJ databases were searched for appropriate papers. The English terms: 'DKK1', 'Dickkopf-1' 'Dickkopf related protein 1', 'psoriatic arthritis' and 'PsA' were used for search purposes. Eight original articles and two reviews were identified up to August 2023. In four out of 8 discussed studies DKK1 serum level was higher in PsA patients than in healthy controls [Dalbeth, p < 0.01; Diani, p < 0.001; Chung, p < 0.01; Abd el Hamid, p < 0.001)], it was comparable in another (Daousiss, p = 0.430) and was lower in two (Fassio2017, p < 0.05; Fassio2019, p < 0.05). In one study, the comparative groups included patients with axial spondyloarthritis, where DKK1 serum levels were lower in PsA groups [Jadon, peripheral PsA, p = 0.01]. The true relative serum concentration of DKK1 in PsA, as well as its influence on osteogenesis and osteoresorption, is still equivocal. Further studies on this matter with consistent and stringent methodology are warranted.

Acquired drug resistance interferes with the susceptibility of prostate cancer cells to metabolic stress.

BACKGROUND: Metformin is an inhibitor of oxidative phosphorylation that displays an array of anticancer activities. The interference of metformin with the activity of multi-drug resistance systems in cancer cells has been reported. However, the consequences of the acquired chemoresistance for the adaptative responses of cancer cells to metformin-induced stress and for their phenotypic evolution remain unaddressed. METHODS: Using a range of phenotypic and metabolic assays, we assessed the sensitivity of human prostate cancer PC-3 and DU145 cells, and their drug-resistant lineages (PC-3_DCX20 and DU145_DCX20), to combined docetaxel/metformin stress. Their adaptation responses have been assessed, in particular the shifts in their metabolic profile and invasiveness. RESULTS: Metformin increased the sensitivity of PC-3 wild-type (WT) cells to docetaxel, as illustrated by the attenuation of their motility, proliferation, and viability after the combined drug application. These effects correlated with the accumulation of energy carriers (NAD(P)H and ATP) and with the inactivation of ABC drug transporters in docetaxel/metformin-treated PC-3 WT cells. Both PC-3 WT and PC-3_DCX20 reacted to metformin with the Warburg effect; however, PC-3_DCX20 cells were considerably less susceptible to the cytostatic/misbalancing effects of metformin. Concomitantly, an epithelial-mesenchymal transition and Cx43 upregulation was seen in these cells, but not in other more docetaxel/metformin-sensitive DU145_DCX20 populations. Stronger cytostatic effects of the combined fenofibrate/docetaxel treatment confirmed that the fine-tuning of the balance between energy supply and expenditure determines cellular welfare under metabolic stress. CONCLUSIONS: Collectively, our data identify the mechanisms that underlie the limited potential of metformin for the chemotherapy of drug-resistant tumors. Metformin can enhance the sensitivity of cancer cells to chemotherapy by inducing their metabolic decoupling/imbalance. However, the acquired chemoresistance of cancer cells impairs this effect, facilitates cellular adaptation to metabolic stress, and prompts the invasive front formation.

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

CD44+ cells determine fenofibrate-induced microevolution of drug-resistance in prostate cancer cell populations.

Combinations of metabolic blockers (incl. fenofibrate) with chemotherapeutic drugs interfere with the drug-resistance of prostate cancer cells. However, their effect on cancer stem cells-dependent microevolution of prostate cancer malignancy remains unaddressed. Here, we hypothesize that the combined docetaxel/fenofibrate treatment prompts the selective expansion of cancer stem cells that affects the microevolution of their progenies. Accordingly, we adapted a combined in vitro/in vivo approach to identify biological and therapeutic consequences of this process. Minute subpopulations of docetaxel-resistant CD133high and/or CD44high cancer stem cell-like (SCL) cells were found in prostate cancer DU145 and PC3 cell populations. When pretreated with docetaxel, they readily differentiated into docetaxel-resistant CD44negative "bulk" cells, thus accounting for the microevolution of drug-resistant cell lineages. Combined docetaxel/fenofibrate treatment induced the generation of poly(morpho)nuclear giant cells and drug-resistant CD44high SCL cells. However, the CD44negative offspring of docetaxel- and docetaxel/fenofibrate-treated SCLs remained relatively sensitive to the combined treatment, while retaining enhanced resistance to docetaxel. Long-term propagation of drug-resistant SCL-derived lineages in the absence of docetaxel/fenofibrate resulted in their reverse microevolution toward the drug-sensitivity and invasive phenotype. Consequently, prostate tumors were able to recover from the combined docetaxel/fenofibrate stress after the initial arrest of their expansion in vivo. In conclusion, we have confirmed the potential of fenofibrate for the metronomic treatment of drug-resistant prostate tumors. However, docetaxel/fenofibrate-induced selective expansion of hyper-resistant CD44high SCL prostate cells and their "bulk" progenies prompts the microevolution of prostate tumor drug-resistance. This process can limit the implementation of metabolic chemotherapy in prostate cancer treatment.

WNT/ß-Catenin Signaling Promotes TGF-ß-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production.

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-ß and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-ß-activated human cardiac fibroblasts. We found that WNT3a and TGF-ß induced a ß-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-ß triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the ß-catenin pathway with the GSK3ß inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-ß, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-ß-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-ß and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-ß-induced production of collagen I. In conclusion, WNT/ß-catenin signaling promotes TGF-ß-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.

Role of HMOX1 Promoter Genetic Variants in Chemoresistance and Chemotherapy Induced Neutropenia in Children with Acute Lymphoblastic Leukemia.

Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(-413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.

The specific ex vivo released cytokine profile is associated with ischemic stroke outcome and improves its prediction.

BACKGROUND: Inflammation is associated with poor outcome after stroke. A relationship between ex vivo cytokine synthesis and stroke outcome remains unclear. We explored an association between ex vivo cytokine release, circulating interleukin (IL)-6 as a marker of systemic inflammation, and stroke prognosis. We assessed the utility of ex vivo synthesized cytokines for predicting stroke outcome. METHODS: We collected blood from 248 ischemic stroke patients and stimulated it ex vivo with lipopolysaccharide. We measured concentration of synthesized cytokines (TNFα, IP-10, IL-1ß, IL-6, IL-8, IL-10, and IL-12) and plasma IL-6. We assessed functional outcome 3 months after stroke using the modified Rankin Scale. To assess the prognostic ability of cytokines, we applied multivariate logistic regression, cluster analysis, and construction of multimarker score. RESULTS: Decreased release of IP-10, TNFα, IL-1ß, and IL-12; increased release of IL-10 and IL-8; and higher plasma IL-6 level were associated with poor outcome. Cluster analysis identified three groups of patients with distinct cytokine profiles. The group with the worst outcome demonstrated high synthesis of IL-10, IL-8, IL-1ß, and IL-6 and low synthesis of IL-12, IP-10, and TNFα accompanied by high circulating IL-6 level. The group with the best prognosis showed high synthesis of TNFα, IP-10, IL-12, IL-1ß, and IL-6; low synthesis of IL-10 and IL-8; and low plasma IL-6. Patients with intermediate outcome had low synthesis of all cytokines accompanied by low circulating IL-6. We constructed a multimarker score composed of ex vivo released IL-12, IL-10, TNFα, and plasma IL-6. Addition of this score to clinical variables led to significant increase in c-statistic (0.81 vs 0.73, p = 0.02) and net reclassification improvement. CONCLUSION: The decreased ex vivo release of pro-inflammatory cytokines and increased release of IL-10 and IL-8 are related to poor outcome after stroke. Cytokine-based multimarker score adds prognostic value to clinical model for predicting stroke outcome.

Decreased number of regulatory T lymphocytes is related to inflammation and number of CD8+ T cells expressing programmed cell death protein-1 in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder related to recurrent infections, as well as a range of non-infectious manifestations including autoimmune and inflammatory disorders. We hypothesized that patients with CVID and different clinical phenotypes would demonstrate alterations in lymphocyte T subsets, including T lymphocytes expressing programmed cell death protein 1 (PD-1), and regulatory T lymphocytes. We performed flow cytometry in two CVID groups: group 1 with infections only, and group 2 with infections and concomitant noninfectious manifestations. Patients were 18-59 years old (mean 35.8 years of age). Increased proportions of CD8+PD-1+ T cells and reduced regulatory T cells were associated with lymphadenopathy. Amount of regulatory T cells correlated with CD8+PD-1+ T lymphocytes (r = 0.54; p = 0.013), and with CRP (r = -0.64; p = 0.004). Forty percent of patients expressed manifestations in addition to infections (group 2), and they had reduction in number of regulatory T cells [8 (3-12) vs. 24 (11-26)/µl; p = 0.034), naive CD4+ T lymphocytes [36 (27-106) vs. 149 (81-283)/µl; p = 0.034], and elevated C-reactive protein (CRP) [5.33 (3.15-8.82) vs. 1 (1-2.16) mg/l; p = 0.003] in comparison to group 1. In conclusion, the amount of CD8+ T cells expressing PD-1 is associated with lymphadenopathy and number of regulatory T cells in patients with CVID. Patients with CVID and non-infectious complications have increased level of inflammation and alterations in regulatory T cells.

Secondary immunodeficiencies with predominant antibody deficiency: multidisciplinary perspectives of Polish experts.

At present, secondary immune deficiencies have become a clinical problem, recognized in different specialties. The aim of this paper was to increase awareness and support the need for screening at-risk populations. Secondary immune deficiencies result in variety of conditions, but not all of them require immunoglobulin replacement therapy, as specific antibody response might be preserved. Moreover, the management of secondary immune deficiencies vary between countries and different medical disciplines. This literature review presents the most common causes and clinical presentation of secondary immunodeficiencies with predominant impaired antibody production. We present diagnostic guidelines for patients at-risk, with an emphasis on the role of prophylactic vaccination as a treatment and diagnostic tool. This review considers the specificity and disparities of the Polish healthcare system and ultimately, suggests that management teams should include a clinical immunologist experienced in the treatment of humoral immunodeficiencies.

WNT3a and WNT5a Transported by Exosomes Activate WNT Signaling Pathways in Human Cardiac Fibroblasts.

WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20⁻150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3ß (GSK3ß), induced nuclear translocation of ß-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/ß-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate ß-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.

CA-170 - A Potent Small-Molecule PD-L1 Inhibitor or Not?

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins - important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 - a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.

T Regulatory CD4+CD25+FoxP3+ Lymphocytes in the Peripheral Blood of Left-Sided Colorectal Cancer Patients.

Background and objectives: T regulatory lymphocytes (Treg) are one of the subsets of T-lymphocytes involved in the interaction of neoplastic tumors and the host immune system, and they may impair the immune reaction against cancer. It has been shown that Treg are increased in the peripheral blood of patients with various cancers. In colorectal cancer, the prognostic role of Treg remains controversial. Colorectal cancer is a heterogenous disease, with many variations stemming from its primary tumor location. The aim of this study is to analyse the relationship between the amount of Treg in the peripheral blood of patients with left-sided colorectal cancer in various stages of disease and long-term survival. Materials and Methods: A prospective analysis of 94 patients with left-sided colorectal cancer and a group of 21 healthy volunteers was carried out. Treg levels in peripheral blood were analysed using flow cytometry. Results: There was a statistically significant difference between the amount of Treg in the Ist and IInd TNM stages (p = 0.047). The number of Treg in the entire study group was significantly lower than in the control group (p = 0.008) and between patients in stages II and III and the control group (p = 0.003 and p = 0.018). The group of pT3+pT4 patients also had significantly lower Treg counts in their peripheral blood than the control group (p = 0.005). In the entire study group, the level of Treg cells in the peripheral blood had no influence on survival. The analysis of the TNM stage subgroups also showed no difference in survival between patients with "low" and "high" Treg counts. Conclusion: The absolute number of Treg in the peripheral blood of patients with left-sided colorectal cancer was significantly decreased in comparison to healthy controls, especially for patients with stage II+III disease. Treg presence in the peripheral blood had no impact on survival.

Reduced ex vivo release of pro-inflammatory cytokines and elevated plasma interleukin-6 are inflammatory signatures of post-stroke delirium.

BACKGROUND: Experimental studies suggest that systemic inflammation contributes to the pathophysiology of delirium. The aim of our study was to determine blood-derived inflammatory signatures of post-stroke delirium. METHODS: We included 144 ischemic stroke patients. We assessed delirium on a daily basis during the first 7 days of hospitalization. Venous blood was collected at day 3 after the onset of stroke and stimulated ex vivo with lipopolysaccharide (LPS). We measured LPS-induced cytokine concentration (TNFα, IP-10, IL-1ß, IL-6, IL-8, IL-10, and IL-12p70) as well as plasma levels of IL-6 and TNFα. RESULTS: Delirium was diagnosed in 21.5% of patients. After correction for monocyte count, patients with delirium had reduced LPS-induced TNFα, IP-10, IL-1ß, IL-6, and IL-12 release. The plasma IL-6 level was higher in delirious patients compared to patients without delirium. After adjusting for stroke severity and infections, higher ex vivo TNFα (OR 0.29, 95%CI 0.11-0.72, P = 0.01), IP-10 (OR 0.25, 95%CI 0.08-0.73, P = 0.01), IL-1ß (OR 0.42, 95%CI 0.20-0.89, P = 0.02), and IL-12 (OR 0.07, 95%CI 0.01-0.70, P = 0.02) release was associated with the reduced risk of delirium. In multivariate analysis, the higher plasma IL-6 was associated with the increased risk of delirium (OR 1.61, 95%CI 1.00-2.58, P = 0.04). CONCLUSIONS: Reduced ex vivo release of pro-inflammatory cytokines after LPS stimulation and the elevated plasma IL-6 are signatures of post-stroke delirium.

Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro. RESULTS: The human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells. CONCLUSIONS: We designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.